Polynucleotides encoding interleukin-12 (IL12) and uses thereof

Abstract

The present disclosure relates to polynucleotides comprising an open reading frame of linked nucleosides encoding human interleukin-12 (IL12), functional fragments thereof, and fusion proteins comprising IL12. In some embodiments, the open reading frame is sequence-optimized. In particular embodiments, the disclosure provides sequence-optimized polynucleotides comprising nucleotides encoding the polypeptide sequence of human IL12, or sequences having high sequence identity with those sequence optimized polynucleotides.

What is claimed is: 
     
       1. A method for reducing the size of a tumor or inhibiting growth of a tumor in a subject who has received or is receiving an immune checkpoint inhibitor which is an antibody, the method comprising administering to the subject a lipid nanoparticle (LNP) encapsulated messenger RNA (mRNA) comprising an open reading frame (ORF) encoding a fusion protein comprising a human IL-12B polypeptide fused directly or by a linker to a human IL-12A polypeptide, wherein the IL-12B polypeptide comprises the amino acid sequence of SEQ ID NO: 1, wherein the IL-12A polypeptide comprises the amino acid sequence of SEQ ID NO: 3, and wherein the LNP comprises an ionizable amino lipid; a phospholipid; a sterol; and a PEG-modified lipid, thereby reducing the size of the tumor or inhibiting growth of the tumor in the subject. 
     
     
       2. The method of  claim 1 , wherein the ORF comprises from 5′ to 3′ a nucleotide sequence selected from the group consisting of:
 (i) a nucleotide sequence encoding the IL-12B polypeptide, a nucleotide sequence encoding a peptide linker, and a nucleotide sequence encoding the IL-12A polypeptide; 
 (ii) a nucleotide sequence encoding the IL-12B polypeptide, and a nucleotide sequence encoding the IL-12A polypeptide; 
 (iii) a nucleotide sequence encoding the IL-12A polypeptide, a nucleotide sequence encoding a peptide linker, and a nucleotide sequence encoding the IL-12B polypeptide; and 
 (iv) a nucleotide sequence encoding the IL-12A polypeptide, and a nucleotide sequence encoding the IL-12B polypeptide. 
 
     
     
       3. The method of  claim 2 , wherein the ORF comprises a nucleotide sequence encoding a signal peptide located at the 5′ terminus of the ORF. 
     
     
       4. The method of  claim 3 , wherein the signal peptide is a human IL-12B signal peptide. 
     
     
       5. The method of  claim 1 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 48. 
     
     
       6. The method of  claim 1 , wherein the antibody is an anti-PD1 antibody, and anti-PDL1 antibody, or an anti-CTLA4 antibody. 
     
     
       7. A method for reducing the size of a tumor or inhibiting growth of a tumor in a subject who has received or is receiving a LNP encapsulated mRNA comprising an ORF encoding a fusion protein comprising a human IL-12B polypeptide fused directly or by a linker to a human IL-12A polypeptide, wherein the IL-12B polypeptide comprises the amino acid sequence of SEQ ID NO: 1, wherein the IL-12A polypeptide comprises the amino acid sequence of SEQ ID NO: 3, wherein the LNP comprises an ionizable amino lipid; a phospholipid; a sterol; and a PEG-modified lipid, the method comprising administering to the subject an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an antibody, thereby reducing the size of the tumor or inhibiting growth of the tumor in the subject. 
     
     
       8. The method of  claim 7 , wherein the ORF comprises from 5′ to 3′ a nucleotide sequence selected from the group consisting of:
 (i) a nucleotide sequence encoding the IL-12B polypeptide, a nucleotide sequence encoding a peptide linker, and a nucleotide sequence encoding the IL-12A polypeptide; 
 (ii) a nucleotide sequence encoding the IL-12B polypeptide, and a nucleotide sequence encoding the IL-12A polypeptide; 
 (iii) a nucleotide sequence encoding the IL-12A polypeptide, a nucleotide sequence encoding a peptide linker, and a nucleotide sequence encoding the IL-12B polypeptide; and 
 (iv) a nucleotide sequence encoding the IL-12A polypeptide, and a nucleotide sequence encoding the IL-12B polypeptide. 
 
     
     
       9. The method of  claim 8 , wherein the ORF comprises a nucleotide sequence encoding a signal peptide located at the 5′ terminus of the ORF. 
     
     
       10. The method of  claim 9 , wherein the signal peptide is a human IL-12B signal peptide. 
     
     
       11. The method of  claim 7 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 48. 
     
     
       12. The method of  claim 7 , wherein the antibody is an anti-PD1 antibody, and anti-PDL1 antibody, or an anti-CTLA4 antibody. 
     
     
       13. A method for reducing the size of a tumor or inhibiting growth of a tumor in a subject, comprising administering to the subject (i) an immune checkpoint inhibitor, wherein the immune checkpoint inhibitor is an antibody; and (ii) an LNP encapsulated mRNA comprising an ORF encoding a fusion protein comprising a human IL-12B polypeptide fused directly or by a linker to a human IL-12A polypeptide, wherein the IL-12B polypeptide comprises the amino acid sequence of SEQ ID NO: 1, wherein the IL-12A polypeptide comprises the amino acid sequence of SEQ ID NO: 3, wherein the LNP comprises an ionizable amino lipid; a phospholipid; a sterol; and a PEG-modified lipid, thereby reducing the size of the tumor or inhibiting growth of the tumor in the subject. 
     
     
       14. The method of  claim 13 , wherein the immune checkpoint inhibitor and the LNP encapsulated mRNA are administered sequentially. 
     
     
       15. The method of  claim 13 , wherein the LNP encapsulated mRNA is administered prior to administration of the immune checkpoint inhibitor. 
     
     
       16. The method of  claim 13 , wherein the immune checkpoint inhibitor and the LNP encapsulated mRNA are administered concurrently. 
     
     
       17. The method of  claim 13 , wherein the ORF comprises from 5′ to 3′ a nucleotide sequence selected from the group consisting of:
 (i) a nucleotide sequence encoding the IL-12B polypeptide, a nucleotide sequence encoding a peptide linker, and a nucleotide sequence encoding the IL-12A polypeptide; 
 (ii) a nucleotide sequence encoding the IL-12B polypeptide, and a nucleotide sequence encoding the IL-12A polypeptide; 
 (iii) a nucleotide sequence encoding the IL-12A polypeptide, a nucleotide sequence encoding a peptide linker, and a nucleotide sequence encoding the IL-12B polypeptide; and 
 (iv) a nucleotide sequence encoding the IL-12A polypeptide, and a nucleotide sequence encoding the IL-12B polypeptide. 
 
     
     
       18. The method of  claim 17 , wherein the ORF comprises a nucleotide sequence encoding a human IL-12B signal peptide located at the 5′ terminus of the ORF. 
     
     
       19. The method of  claim 13 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 48. 
     
     
       20. The method of  claim 13 , wherein the antibody is an anti-PD1 antibody, and anti-PDL1 antibody, or an anti-CTLA4 antibody. 
     
     
       21. The method of  claim 1 , wherein the antibody is an anti-PDL1 antibody. 
     
     
       22. The method of  claim 7 , wherein the antibody is an anti-PDL1 antibody. 
     
     
       23. The method of  claim 13 , wherein the antibody is an anti-PDL1 antibody.